The present invention relates to a method for assaying human pepsin II or human pepsinogen II in the human body fluid (such as gastric juice, blood, urine etc.) as diagnostic marker of gastric diseases such as gastric cancer, gastric ulcer etc. and a peptide used as a substrate in such a method.
More detailed, the present invention relates to
1) a peptide of the formula (I) 
(wherein, Q is Qaxe2x80x94(AA)nxe2x80x94 (in which AA is L-amino acid, n is 0 or an integer of 1xcx9c15, Qa is hydrogen, C1xcx9c4 alkyl, an amino-protective group, D- or L-amino acid residue or NH2xe2x80x94(CH2)rxe2x80x94COxe2x80x94 (in which r is an integer of 2xcx9c7.).),
R1 and R2 are (i) hydrogen or halogen or, (ii) R1, R2 and unsaturated bond together form an aromatic carbon ring may be substituted by halogen,
R3 is hydrogen or halogen,
R4 is hydrogen, C1xcx9c3 alkyl or hydroxymethyl,
EE is D- or L-amino acid residue,
m is 0 or 1,
Z is an aniline derivative residue, an aminocoumarine derivative residue or an aminonaphthalene derivative residue,
with the proviso that (1) when n is 2 or more, each AA is same or different, and that (2) the compounds wherein all of R1, R2 and R3 is hydrogen are excluded.)
or an acid addition salt thereof and,
2) a method for assaying human pepsinogen II or human pepsin II which is characterized by digesting a peptide of the formula (I) (wherein all the symbols are as defined hereinbefore.) described in the said 1) or an acid addition salt thereof by human pepsin II which is obtained by activation of human pepsinogen II in a sample or human pepsin II in a sample to obtain an amino acid derivative of the formula (II) 
(wherein all the symbols are as defined hereinbefore.),
digesting the obtained amino acid derivative by aminopeptidase to obtain an aniline, aminocoumarine or aminonaphthalene derivative of the formula Zxe2x80x94H and then
detecting the obtained aniline, aminocoumarine or aminonaphthalene derivative and
3) a kit for assaying human pepsinogen II or human pepsin II which is characterized by comprising a peptide of the formula (I) (wherein all the symbols are as defined hereinbefore.) described in the said 1) or an acid addition salt thereof as a substrate and an aminopeptidase.
It is known that the pepsinogen secretion is parallel to gastric acid secretion and that human serum or urine pepsinogen levels are also parallel to gastric pepsinogen secretion. The above pepsinogen exists as pepsinogen in the body fluid such as blood or urine except for gastric juice, on the other hand, it exists as pepsin in gastric juice.
It is said that human blood or urine pepsinogen I level of the patient with atrophic gastritis decreases and that human blood or urine pepsinogen I and pepsinogen II levels increase in case of gastric ulcer (Japanese Patent Application Kokai Hei 7-304800). In addition, it is said that both pepsinogen I level and pepsinogen II/I ratio decrease in the patient with gastric cancer (Jpn. J. Cancer Res., 80, 111-114 (1989)).
Further, an attention is paid to serum pepsinogen II level and pepsinogen I/II ratio as markers of therapy for helicobacter pylori gastritis. That is to say, it is said that serum pepsinogen II level decreases significantly and pepsinogen I/II ratio increases significantly in a successful group consisting of patients in whom therapy resulted in eradication of the bacteria to compare with an unsuccessful group consisting of patients who remained infected (Prog. Med., 15, 1862-1868 (1995)).
Therefor, assaying the level of human pepsinogen II in human blood or urine may be useful for diagnosis at early stage of diseases such as gastric cancer, gastric ulcer and duodenal ulcer etc.
As for a method for assaying human pepsin which was obtained by activation of human pepsinogen, a method using human serum protein etc. in urine and serum based on its digesting activity has been known (Clin. Chem., 15, 1, 42-55 (1969)). The significance of clinical trial using such a method has been discussed, but it requires a long time. In addition, its accuracy was not good, so such a method has been of no practical use. Further, the results means the activity to digest protein, so it was reflected on the total activities of both pepsin I and pepsin II. Therefore it is impossible to determine the human serum pepsinogen II specifically.
Recently, a method for assaying human pepsinogen in urine (uropepsin) indirectly, based on inactivation of an acidic enzyme by activated pepsin was proposed (Japanese Patent Application Kokai Hei 7-155198). But the substrate used in this method did not show the specificity for pepsin II. It is said that the pepsinogen in urine is pepsinogen I. But, pepsin II may be also secreted in urine in some body condition, so it is difficult to determine the accurate level of pepsin II. It is impossible to assay the level of pepsinogen II in human serum specifically.
As for a method for assaying pepsinogen II, radio immunoassay (Kaku-igaku (in Japanese), Vol. 26, No. 9, 1217-1221 (1989)) and enzyme immunoassay (Japanese Patent Application Kokai Hei 7-304800) using a specific anti-body have been practical use, but these methods cause a radioactive pollution and require a long time and complicated procedure for assaying.
Some methods for assaying pepsin II using synthesized substrate have been proposed. For example, (A) in the paper of J. Med., Chem., 36, 2614 (1993), it was described that a peptide of the formula (A-1)
LysProAlaAlaPhePhe(NO2)ArgLeu xe2x80x83xe2x80x83(A-1) (SEQ ID NO:86)
(wherein Phe(NO2) is p-nitrophenylalanine.)
was used as a substrate in assaying inhibitory effect of some compounds on human pepsin II. That is to say, a peptide of the formula (A-1) was digested by human pepsin II to obtain a peptide of the formula (A-2)
Phe(NO2)ArgLeuxe2x80x83xe2x80x83(A-2)
(wherein Phe(NO2) is as defined hereinbefore.),
and the obtained peptide of the formula (A-2) was used in the assaying inhibitory activity on enzyme based on decrease of absorbance at 234xcx9c324 nm as an index.
But this paper did not disclose that such a peptide of the formula (A-1) may be as a substrate for human pepsin II. Therefore, it is uncertain whether this peptide has specificity for human pepsin II, or not. In addition, the chromophore of this peptide is Phe(NO2), so it is expected that the accuracy of the method using this peptide is one tenth or less to compare with p-nitroaniline (abbreviated as pNA). Further, it is impossible to be used in automated clinical analyzer due to detection at 234xcx9c324 nm.
(B) in the paper of J. Biochem., 265, 871-878 (1990), it was described that a peptide of the formula (B-1)
LysProValValPhePhe(NO2)ArgLeuxe2x80x83xe2x80x83(B-1) (SEQ ID NO:87)
(wherein Phe(NO2) is as defined hereinbefore.),
was used as a substrate in assaying inhibitory effect of some compounds on human pepsin II. That is to say, a peptide of the formula (B-1) was digested by human pepsin II to obtain a peptide of the formula (B-2)
Phe(NO2)ArgLeuxe2x80x83xe2x80x83(B-2)
(wherein Phe(NO2) is as defined hereinbefore.),
and the obtained peptide of the formula (B-2) was used in the assaying inhibitory activity on enzyme based on decrease of absorbance at 234xcx9c324 nm as an index.
But this paper did not disclose that such a peptide of the formula (B-1) may be as a substrate for human pepsin II. Therefore, it is uncertain whether this peptide has a specificity for human pepsin II, or not. In addition, the chromophore of this peptide is Phe(NO2), so it is expected that the accuracy of the method using this peptide is one tenth or less to compare with p-nitroaniline (abbreviated as pNA). Further, it is impossible to be used in automated clinical analyzer due to detection at 234xcx9c324 nm (There are the same problems as in paper (A).).
(C) In CS-261172, it was described that a peptide of the formula (C-1)
Xxe2x80x94Axe2x80x94B-Phe-D-pNAxe2x80x83xe2x80x83(C-1)
(wherein X is hydrogen, C3xcx9c5 carboxylalkylcarbonyl or C1xcx9c5 alkylcarbonyl,
A is pyroglutamic acid (abbreviated as pGlu), Asp, Glu or Gly residue or 2-oxoimidazoline-1-yl-carbonyl,
B is His, Gly or Pro residue,
D is Phe, Leu, Nle, Met or Sxe2x80x94C1xcx9c3 alkyl-Cys residue, and pNA is p-nitroaniline residue.)
was used as a substrate in assaying activities of pepsin I, pepsin II and chymosin.
But, there was neither description nor suggestion about the specificity of this peptide for human pepsin II.
In the example, a peptide of the formula (C-2)
pGluHisPhePhe-pNAxe2x80x83xe2x80x83(C-2) (SEQ ID NO:88)
(wherein pGlu and pNA are as defined hereinbefore.)
was used in assaying activity of pig pepsin. This pig pepsin was not purified and thought to be a mixture of pepsin I and pepsin II. So, it is not expected that this substrate possesses a specificity for human pepsin II.
(D) In the paper of Clinica Chimica Acta 213, 103-110 (1992), it was described that a peptide of the formula (D-1)
Xdxe2x80x94(AAd)nd-Phe-Dd-pNAxe2x80x83xe2x80x83(D-1)
(wherein Xd is pGlu, Glt, AAd is sequence for a substrate for N-termini, nd is 1 or 2, Dd is Phe, Nle or Met and the other symbols are as defined hereinbefore.)
was used as a substrate in assaying activities of pepsin I, pepsin II and chymosin.
But, there was neither description nor suggestion about the specificity of this peptide for human pepsin II.
In the experimental example, a peptide of the formula (D-2) or (D-3)
pGluHisPhePhe-pNA xe2x80x83xe2x80x83(D-2) or (SEQ ID NO:90)
pGluHisPheMet-pNA xe2x80x83xe2x80x83(D-3) (SEQ ID NO:91)
(wherein pGlu and pNA are as defined hereinbefore.)
was used as a substrate in assaying activities of pig pepsin. This pig pepsin was not purified and thought to be a mixture of pepsin I and pepsin II. So, it is not expected that this substrate possesses a specificity for human pepsin II.
(E) In the paper of Anal. Biochem., 234, 113 (1996), it was described that peptides of the formula (E-1) or (E-2)
AbzAlaAlaPhePheAlaAla-Dedxe2x80x83xe2x80x83(E-1), or (SEQ ID NO:92)
AbzAlaAlaPhePheAlaAla-pNAxe2x80x83xe2x80x83(E-2) (SEQ ID NO:93)
(wherein Abz is o-aminobenzoyl, Ded is N-2,4-dinitrophenylethylene-diamine and pNA is as defined hereinbefore.), were used as substrate in assaying activities of human pepsin I, human pepsin II, human cathepsin D and HIV protease by fluorophotometry.
In this paper, the fluorescent changes caused by modification of peptide was discussed. Particularly, a peptide of the formula (E-1) seemed to be suggested to have specificity for human pepsin I from the experimental result. But, the peptides of the formula (E-1) or (E-2) were not expected to have the specificity for human pepsin II.
(F) In the paper of J. Biol. Chem., 244, 5, 1085-1091 (1969), it was described that peptides of the formula (F-1)xcx9c(F-5)
Z-TyrAlaxe2x80x83xe2x80x83(F-1),
Z-TyrThrxe2x80x83xe2x80x83(F-2),
Z-TyrLeuxe2x80x83xe2x80x83(F-3),
Z-TyrSerxe2x80x83xe2x80x83(F-4) or
Z-TrpAlaxe2x80x83xe2x80x83(F-5)
(wherein Z is benzyloxycarbonyl.)
were used as substrate in assaying activities of human pepsin II specifically. But it requires a long time and complicated procedure. In addition, its accuracy was not good, so such a method has been of no practical use.
The substrates described in the said five references (A)xcx9c(F) are distinct from the substrate (peptide) of the present invention entirely. That is to say, there is a structural characteristic that an amino acid of the formula 
(wherein all the symbols are as defined hrereinbefore.) in the peptide of the formula (I) of the present invention is not an essential amino acid (for example, Phe, Tyr) but phenylalanine substituted by halogen or 2-naphthylalanine (in which naphthyl may be substituted by halogen.). Such a group was neither described nor suggested in the said six references (A)xcx9c(F).
The present inventors have been studying to dissolve these problems of the related arts and to find a substrate which is high-sensitive (being high rate of enzyme reaction i.e., digesting a substrate by human pepsin II at a high rate and/or being able to produce efficient coloring) and specific for human pepsin II, and then have succeeded in synthesizing a substrate (peptide) which is sensitive and specific for human pepsin II. By using this substrate, it become to possible to determine pepsin II for short time to compare with the method of related arts and to determine pepsinogen II and pepsin II using automated clinical analyzer.
That is to say, the present invention relates to
1) a peptide of the formula (I) 
(wherein all the symbols are as defined hereinbefore.), or acid addition salt thereof,
2) a method for assaying human pepsinogen II or human pepsin II characterized by
digesting a peptide of the formula (I) described in the above 1) (wherein all the symbols are as defined hereinbefore.), or an acid addition salt thereof, by human pepsin II which is obtained by activation of human pepsinogen II in a sample or human pepsin II in a sample to obtain an amino acid derivative of the formula (II) 
(wherein all the symbols are as defined hereinbefore.),
digesting the obtained amino acid derivative by aminopeptidase to obtain an aniline, aminocoumarine or aminonaphthalene derivative of the formula Zxe2x80x94H and then
detecting the obtained aniline, aminocoumarine or aminonaphthalene derivative,
3) a kit for assaying human pepsinogen II or human pepsin II which is characterized by comprising a peptide of the formula (I) described in the above 1), or an acid addition salt thereof as a substrate and aminopeptidase.